BE115+-+Lab01 - Lab 1: Cell Culture 1 Introduction The use...

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Page 1 of 11 Bioengineering 115: Cell Biology for Engineers Laboratory 1: Cell Culture Last updated: January 31, 2011 Lab 1: Cell Culture 1 Introduction The use of cultured cells grown in vitro has several advantages over the use of an in vivo system or an isolated tissue preparation in an experimental approach. This technique allows the researcher to easily manipulate specific aspects of the cellular environment and prevents exposure of an organism to toxic and other pharmacological agents. Furthermore, the experimenter can selectively alter and study one variable at a time without inducing secondary or tertiary effects of another system. Adding a chemical or growth factor to a dish or flask of cells to determine its effect on those cells is experimentally superior (in some ways) to simply injecting that factor or chemical into the body. In the body, it is difficult to separate the direct effects of the factor or chemical on the cells to the indirect effects. Say you add factor X to cell type A in a dish, and observe the effects. At the same time, you inject factor X into a lab animal, sacrifice the animal, and observe type A cells. With the injection, all of the following (and more) are possible. Factor X acts directly on cell type A. Factor X effects cell type B, which releases factor Y, which then effects cell type A. Factor X does nothing, because factor X is taken up by the liver before it can act on cell type A. Factor X does nothing, because it genuinely has no effect on cell type A. When you use an isolated cell line, you remove a number of variables that make your results more difficult to interpret. At the same time, your experiment becomes specific to a particular cell, excluding practical contributions from the cell’s natural environment (the body). Most cell types are grown as a single thickness cell layer (monolayer) or sheet attached to glass or tissue culture treated plastic substrates. In order to keep a culture actively growing, it is necessary to subdivide (e.g., split or passage) them at regular intervals. Passaging initially involves the breakage of intercellular and cell-to-substrate connections by the use or proteolytic enzymes (trypsin, collagenase, etc.). After the cells have been disassociated into a suspension consisting of single cells, they are counted, diluted, and transferred into fresh culture vessels. They re-attach, begin to grow and divide and after a period of incubation (depending on the initial cell number, growth conditions, and the cell line) they reach confluency. A passage of cells is considered confluent when the cells cover the entire surface, and there is no room left for new cells. At this point the cells must be passaged and subcultured (a portion of the cells used to create a new culture) again. When dealing with cultured cells, it’s important to keep an eye on the growth media’s
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BE115+-+Lab01 - Lab 1: Cell Culture 1 Introduction The use...

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